Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker |
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Authors: | Kazuhito Akama Holger Puchta Barbara Hohn |
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Institution: | (1) Friedrich-Mieseher-Institut, P.O. Box 2543, CH-4002 Basel, Switzerland;(2) Present address: Department of Biology, Shimane University, 690 Matsue, Japan |
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Abstract: | Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations
bar
bialaphos resistance gene
- CIM
callus-inducing medium
- DTNB
5,5-dithiobis(2-nitrobenzoic acid)
- GM
germination medium
- HPT
hygromycin phosphotransferase
- MS
Murashige and Skoog salts
- NPTII
neomycin phosphotransferase II
- PAT
phosphinothricin acetyltransferase
- PPT
phosphinothricin
- SIM
shoot-inducing medium |
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Keywords: | Arabidopsis thaliana Transformation Phosphinothricin Bar Basta resistance |
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